PSX-B-16 - Recovery of live cells from refrigerated sheep skin after different days of cold storage

Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2-3 mm²) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO₂ incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with >80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.